Cytosolic galectin-4 enchains bacteria, restricts their motility, and promotes inflammasome activation in intestinal epithelial cells.

Galectin-4, a member of the galectin family of animal glycan-binding proteins (GBPs), is specifically expressed in gastrointestinal epithelial cells and is known to be able to bind microbes. However, its function in host-gut microbe interactions remains unknown. Here, we show that intracellular galectin-4 in intestinal epithelial cells (IECs) coats cytosolic Salmonella enterica serovar Worthington and induces the formation of bacterial chains and aggregates. Galectin-4 enchains bacteria during their growth by binding to the O-antigen of lipopolysaccharides. Furthermore, the binding of galectin-4 to bacterial surfaces restricts intracellular bacterial motility. Galectin-4 enhances caspase-1 activation and mature IL-18 production in infected IECs especially when autophagy is inhibited. Finally, orally administered S. enterica serovar Worthington, which is recognized by human galectin-4 but not mouse galectin-4, translocated from the intestines to mesenteric lymph nodes less effectively in human galectin-4-transgenic mice than in littermate controls. Our results suggest that galectin-4 plays an important role in host-gut microbe interactions and prevents the dissemination of pathogens. The results of the study revealed a novel mechanism of host-microbe interactions that involves the direct binding of cytosolic lectins to glycans on intracellular microbes.

Galectin-3 promotes noncanonical inflammasome activation through intracellular binding to lipopolysaccharide glycans.

Cytosolic lipopolysaccharides (LPSs) bind directly to caspase-4/5/11 through their lipid A moiety, inducing inflammatory caspase oligomerization and activation, which is identified as the noncanonical inflammasome pathway. Galectins, ß-galactoside-binding proteins, bind to various gram-negative bacterial LPS, which display ß-galactoside-containing polysaccharide chains. Galectins are mainly present intracellularly, but their interactions with cytosolic microbial glycans have not been investigated. We report that in cell-free systems, galectin-3 augments the LPS-induced assembly of caspase-4/11 oligomers, leading to increased caspase-4/11 activation. Its carboxyl-terminal carbohydrate-recognition domain is essential for this effect, and its N-terminal domain, which contributes to the self-association property of the protein, is also critical, suggesting that this promoting effect is dependent on the functional multivalency of galectin-3. Moreover, galectin-3 enhances intracellular LPS-induced caspase-4/11 oligomerization and activation, as well as gasdermin D cleavage in human embryonic kidney (HEK) 293T cells, and it additionally promotes interleukin-1ß production and pyroptotic death in macrophages. Galectin-3 also promotes caspase-11 activation and gasdermin D cleavage in macrophages treated with outer membrane vesicles, which are known to be taken up by cells and release LPSs into the cytosol. Coimmunoprecipitation confirmed that galectin-3 associates with caspase-11 after intracellular delivery of LPSs. Immunofluorescence staining revealed colocalization of LPSs, galectin-3, and caspase-11 independent of host N-glycans. Thus, we conclude that galectin-3 amplifies caspase-4/11 oligomerization and activation through LPS glycan binding, resulting in more intense pyroptosis-a critical mechanism of host resistance against bacterial infection that may provide opportunities for new therapeutic interventions.

Galectin-9 Is Critical for Mucosal Adaptive Immunity through the T Helper 17-IgA Axis.

Impairment of the intestinal mucosal immunity significantly increases the risk of acute and chronic diseases. IgA plays a major role in humoral mucosal immunity to provide protection against pathogens and toxins in the gut. Here, we investigated the role of endogenous galectin-9, a tandem repeat-type ß-galactoside-binding protein, in intestinal mucosal immunity. By mucosal immunization of Lgals9-/- and littermate control mice, it was found that lack of galectin-9 impaired mucosal antigen-specific IgA response in the gut. Moreover, Lgals9-/- mice were more susceptible to developing watery diarrhea and more prone to death in response to high-dose cholera toxin. The results indicate the importance of galectin-9 in modulating intestinal adaptive immunity. Furthermore, bone marrow chimera mice were established, and galectin-9 in hematopoietic cells was found to be critical for adaptive IgA response. In addition, immunized Lgals9-/- mice exhibited lower expression of Il17 and fewer T helper 17 (Th17) cells in the lamina propria, implying that the Th17-IgA axis is involved in this mechanism. Taken together, these findings suggest that galectin-9 plays a role in mucosal adaptive immunity through the Th17-IgA axis. By manipulating the expression or activity of galectin-9, intestinal mucosal immune response can be altered and may benefit the development of mucosal vaccination.

The tuberous sclerosis complex-mammalian target of rapamycin pathway maintains the quiescence and survival of naive T cells.

Naive T cells receive stimulation from the positive selecting ligand in the periphery for their survival. This stimulation does not normally lead to overt activation of T cells, as the T cells remain largely quiescent until they receive either antigenic or lymphopenic stimuli. The underlying mechanism responsible for survival and quiescence of the naive T cells remains largely unknown. In this study, we report that T cell-specific deletion of Tsc1, a negative regulator of mammalian target of rapamycin, resulted in both spontaneous losses of quiescence and cellularity, especially within the CD8 subset. The Tsc1-deficient T cells have increased cell proliferation and apoptosis. Tsc1 deletion affects the survival and quiescence of T cells in the absence of antigenic stimulation. Loss of quiescence but not cellularity was inhibited by rapamycin. Our data demonstrate that tuberous sclerosis complex-mammalian target of rapamycin maintains quiescence and survival of T cells.

Galectin-8 Is Upregulated in Keratinocytes by IL-17A and Promotes Proliferation by Regulating Mitosis in Psoriasis.

Psoriasis is a chronic inflammatory skin disease that develops under the influence of the IL-23/T helper 17 cell axis and is characterized by intense inflammation and prominent epidermal hyperplasia. In this study, we demonstrate that galectin-8, a ß-galactoside‒binding lectin, is upregulated in the epidermis of human psoriatic skin lesions as well as in a mouse model of psoriasis induced by intradermal IL-23 injections and in IL-17A‒treated keratinocytes. We show that keratinocyte proliferation is less prominent in galectin-8‒knockout mice after intradermal IL-23 treatment than in wild-type mice. In addition, we show that galectin-8 levels in keratinocytes are positively correlated with the ability of the cells to proliferate and that transitioning from mitosis into G1 phase is delayed in galectin-8‒knockout HaCaT cells after cell-cycle synchronization and release. We demonstrate by immunofluorescence staining and immunoblotting the presence of galectin-8 within the mitotic apparatus. We reveal by coimmunoprecipitation and mass spectrometry analysis that α-tubulin interacts with galectin-8 during mitosis. Finally, we show that in the absence of galectin-8, pericentrin compactness is lessened and mitotic microtubule length is shortened, as demonstrated by immunofluorescence staining. We conclude that galectin-8 is upregulated in psoriasis and contributes to the hyperproliferation of keratinocytes by maintaining centrosome integrity during mitosis through interacting with α-tubulin.

CD24-p53 axis suppresses diethylnitrosamine-induced hepatocellular carcinogenesis by sustaining intrahepatic macrophages.

It is generally assumed that inflammation following diethylnitrosamine (DEN) treatment promotes development of hepatocellular carcinoma (HCC) through the activity of intrahepatic macrophages. However, the tumor-promoting function of macrophages in the model has not been confirmed by either macrophage depletion or selective gene depletion in macrophages. Here we show that targeted mutation of Cd24 dramatically increased HCC burden while reducing intrahepatic macrophages and DEN-induced hepatocyte apoptosis. Depletion of macrophages also increased HCC burden and reduced hepatocyte apoptosis, thus establishing macrophages as an innate effector recognizing DEN-induced damaged hepatocytes. Mechanistically, Cd24 deficiency increased the levels of p53 in macrophages, resulting in their depletion in Cd24-/- mice following DEN treatment. These data demonstrate that the Cd24-p53 axis maintains intrahepatic macrophages, which can remove hepatocytes with DNA damage. Our data establish a critical role for macrophages in suppressing HCC development and call for an appraisal of the current dogma that intrahepatic macrophages promote HCC development.

Sodium arsenite inhibits migration of extravillous trophoblast cells in vitro.

This study used a first-trimester human extravillous trophoblast (EVT) cell line, HTR-8/SVneo, to investigate whether sodium arsenite (AsNaO(2)) reduces human EVT migration and invasion. Treatments with 2.5 microM AsNaO(2) or less (< or =187.3 microg/L), concentrations that are relevant to human exposures in drinking water, were sublethal to HTR-8/SVneo cells. A 72-h exposure to sodium arsenite inhibited cell migration in a concentration-dependent manner at 0.625, 1.25 and 2.5 microM. Significant changes in cell proliferation were not observed under these treatment conditions. Moreover, inhibition of cell migration was unrelated to phosphorylation of focal adhesion kinase Tyr397. In contrast to cell migration, 72-h exposures to AsNaO(2) (0.3125-2.5 microM) had no significant effects on cell invasion, nor on the activities and protein expression of matrix metalloproteinase (MMP) 2 and MMP9. Because trophoblast migration is important for placentation, these results suggest an effect that could contribute to insufficiency of placental development and adverse pregnancy outcomes.

Trap1a is an X-linked and cell-intrinsic regulator of thymocyte development.

The X-linked Trap1a gene encodes the tumor rejection antigen P1A, which is expressed in fetal tissues and multiple lineages of tumor cells. The function of this gene remains unknown. Using chimeric mice with wild-type (WT) and Trap1a-/y bone marrow, we show that Trap1a-/y donor cells are capable of generating most lineages of hematopoietic cells, with the notable exception of T cells. Deletion of Trap1a selectively arrests T-cell development at double-negative stage 1 (DN1, with a CD4-CD8-CD25-CD44+ phenotype). Because Trap1a is expressed in Lin-Sca-1+c-Kit+ and common lymphoid progenitors but not in immature thymocytes (DN1-DN4), Trap1a mutations affect the differentiation potential of progenitor cells without directly acting on T cells. Despite a similarity in the blockade of DN1 to DN2 transition, the Trap1a-/y DN1 cells have normal expression of c-Kit, in contrast to what was reported in the Notch1-/- DN1. Complementary DNA profiling of Trap1a-/y and WT embryonic stem cells shows that Trap1a does not regulate the Notch pathway. Our data reveal that Trap1a is an X-linked regulator that affects the differentiation potential of progenitor cells into T cells through a Notch-independent mechanism and identify an important function for the Trap1a gene.

Examination of galectins in phagocytosis.

Galectins, a family of ß-galactoside-binding proteins, are expressed in many different phagocytic leukocytes (granulocytes, monocytes, and macrophages). A number of family members have been shown to play an important role in ingestion of particles (phagocytosis), thus contributing to clearance of damaged cells and host defense against pathogens. Here we describe procedures for analysis of the roles of galectins in phagocytosis by using galectin-3 as an example. We emphasize the function of endogenous galectin-3 as determined by comparison of phagocytosis by macrophages from galectin-3 knockout mice and wild-type mice. We focus on the role of galectin-3 in phagocytosis of pathogens and Fcγ receptor-mediated phagocytosis of opsonized cells and particles.

Transgenic expression of P1A induced thymic tumor: a role for onco-fetal antigens in tumorigenesis.

P1A is the first known tumor rejection antigen. It is expressed in embryonic stem cells and multiple tumors but is silent in adult tissues except for the testis and placenta. Therefore, P1A represents a prototype for onco-fetal antigens. To test the potential function of P1A in tumorigenesis, we used a transgenic mouse expressing P1A in lymphoid cells. We observed that immunodeficient host P1A transgenic mice developed thymic tumors after 7 months of age and had shorter survival rates compared to control groups. Most of the 7 examined tumors displayed B cell lineage markers. The P1A transgenic bone marrow cells had higher proliferation ability and more potential progenitors compared to control bone marrow cells. To our knowledge, our data provided the first example that onco-fetal antigen can promote tumorigenesis.

Analysis of oxidative DNA damage 8-hydroxy-2'-deoxyguanosine as a biomarker of exposures to persistent pollutants for marine mammals.

An oxidative DNA biomarker, 8-hydroxy-2'-deoxyguanosine (8-OHdG), was determined in the livers and kidneys of stranded or by-caught cetaceans along the Taiwan coast through isotope-dilution liquid chromatography coupled with tandem mass spectrometry (LC/MS/MS) to evaluate the feasibility of analyzing the DNA adduct in marine mammals and then to study the association between 8-OHdG and levels of polychlorinated biphenyls (PCBs) and dichlorodiphenyldichloroethylene (DDE) in the blubbers of the cetaceans. The mean values of the 8-OHdG from the liver and kidney samples were 19.83+/-10.00 pmol/micromol deoxyguanosine (dG) (6.90-53.53 pmol/micromol dG) and 19.16+/-7.48 pmol/micromol dG (5.36-39.36 pmol/micromol dG), respectively. In general, 8-OHdG was not related to the general health status of the by-caught and stranded animals and also was not related to species. However, the levels of 8-OHdG had a positive correlation with concentrations of PCBs, but not DDE, in female cetacean livers. In addition, when selected coplanar PCBs (dioxin-like congeners) were used to compare the 2,3,7,8-tetrachlorodibenzo-p-dioxin equivalents (TEQs) with 8-OHdG of by-caught cetaceans, a high positive correlation (r = 0.80, p < 0.01) was found in mature female animals. Thus, the detection of 8-OHdG in marine mammals with isotope-dilution LC/MS/ MS is possible, and the study of the relationship between oxidative DNA damage and environmental contaminants under natural exposure indicates that the level of 8-OHdG in female cetacean livers is associated with coplanar PCBs and the factor of sexual maturity.